臺灣博碩士論文加值系統

在一般未受傷的血管壁內，平滑肌細胞主要是以收縮型的方式存在。在動脈硬化形成的過程中，平滑肌細胞則會轉變成合成型的細胞，開始增生並且向血管內層遷移。單核球趨化蛋白-1 (monocyte chemotactic protein-1) 為極佳的單核球趨化物，在血管病變的過程中表現量有顯著增加的情形，因此被認為與動脈硬化的形成有關。最近的研究報告指出，單核球趨化蛋白-1在約10-9 M即可引發人類血管內皮細胞的趨化性，然而單核球趨化蛋白-1對於平滑肌細胞的想目前仍不清楚。
在本實驗中，我們以大白鼠胚胎的主動脈平滑肌細胞株A7r5來探討單核球趨化蛋白-1在平滑肌細胞增生與移形作用中所扮演的角色。將MCP-1 cDNA嵌入表現形質體pGEX-2T中，並且送入表現形宿主E.coli BL21中，在利用glutathion sepharose affinity chromatography自BL21中誘導glutathione-S-transferase (GST)-單核球趨化蛋白(GST-MCP-1)融合蛋白表現並純化。我們的實驗結果發現，0.7 nM GST-MCP-1在96小時的連續處理中對於細胞增生並沒有顯著的影響。另一方面，利用已塗上膠原蛋白第一型的濾膜，以modified Boyden chamber測定細胞移行作用，結果顯示GST-MCP-1明顯增加細胞遷移的能力。隨著GST-MCP-1融合蛋白濃度的提高，平滑佳的遷移數目有逐漸增加的趨勢，在0.7 nM達到最大值，約為對照組的1.6倍，且在A7r5細胞株與大白鼠主動脈平滑肌細胞中所得到的結果相似，而GST-MCP-1融合蛋白刺激平滑佳細胞遷移的最佳作用時間為6-8小時.而GST蛋白則無促進平滑肌細胞遷徙的效果。另外，我們以棋盤法分析GST-MCP-1融合蛋白以起平滑肌細胞株的遷移反應，分析後發現GST-MCP-1融合蛋白為平滑肌的趨化物質。此外，NAD(P)H氧化抑制劑diphenyleneiodonium chloride (DPI; 10M) 與superoxide dismutase (SOD; 1U/ml) 的處理可降低由GST-MCP-1融合蛋白所引起的平滑肌細胞的遷移反應，這表示O2- 可能參與在此訊息傳導路徑中。Rho kinase 抑制劑Y27632對GST-MCP-1融合蛋白所引起的平滑肌細胞的遷移反應降低約93%，而不論有無GST-MCP-1融合蛋白的刺激，Erk1/2激抑制劑U0126及p38 MAPK抑制劑SB203580皆可抑制平滑肌細胞發生遷徙現象。另一方面，以GST-MCP-1刺激A7r5細胞會引發Erk1/2的磷酸化，U0126的前處理，則可將此反應完全抑制。由這些結果顯示，GST-MCP-1融合蛋白為平滑肌細胞的趨化物質，而氧活化中間產物、Rho kinase Erk1/2 及p38 MAPK皆參與在GST-MCP-1融合蛋白所引發平滑肌細胞遷移反應的訊息傳遞路徑中。

In the arterial wall, smooth muscle cells (SMCs) normally exist in a quiescent, differentiated state, referred to as the contractile phenotype. During the development of atherosclerosis, SMCs change towards the synthetic phenotype which are capable of proliferation and migration. Monocyte chemotactic protein-1 (MCP-1), a potent monocyte attractant, is markedly upregulated during vascular lesion formation and has been suggested to promote atherogenesis. MCP-1 was recently reported to induce chemotaxis of human endothelial cells at nanomolar concentration. The effects of MCP-1 on vascular SMC functions are still unclear, however.
In this study, we investigated the role of MCP-1 in proliferation and migration of rat embryonic aortic smooth muscle cell line A7r5. The cDNA encoding human MCP-1 was inserted into pGEX-2T expression vector and used to transform an E.coli strain BL21. The fusion protein of glutathione-S-transferase(GST)-MCP-1, was induced and purified from BL21 using glutathione sepharose affinity chromatography. GST-MCP-1 at 0.7 nM exhibited no significant effect on cell proliferation during a 96-h experimental period. On the other hand, GST-MCP-1 significantly increased cell migration activity as measured by modified Boyden chamber method using a polycarbonate membrane coated with type I collagen. The stimulatory effect of GST-MCP-1 on cell migration was biphasic with 10-9 M being maximally effective, approximately 1.6 fold over basal migration. GST-MCP-1 at 0.7 nM exhibited similar stimulation on A7r5 cells and rat aortic SMCs from primary culture. GST alone had no effect on cell migration. GST-MCP-1-induced smooth muscle cell migration peaked at 6-8 hour incubation period. Checkerboard analysis showed that the response of SMC to GST-MCP-1 was chemotactic. Moreover, the NAD(P)H oxidase inhibitor diphenyleneiodonium chloride (10 M) and superoxide dismutase (1 U/ml) diminished GST-MCP-1-induced SMC migration, suggesting that O2- might be involved in the signaling pathway. The Rho kinase inhibitor Y27632 (10-5 M) reduced GST-MCP-1-induced SMC migration by 93%. The extracellular signal-regulated protein kinase 1/2 (ERK1/2) kinase inhibitor U0126 (10 M) and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (50 M) abrogated both the basal migration and GST-MCP-1-induced migration. Moreover, exposure of A7r5 cells to GST-MCP-1 caused rapid phosphorylation of ERK1/2 and this effect was abolished by U0126. These results demonstrate that GST-MCP-1 is a chemoattractant for SMC and that reactive oxygen species, Rho kinase, ERK1/2 and p38 MAPK are involved in GST-MCP-1-stimulated SMC migration.

中文摘要…………………………………………………………………I
英文摘要………………………………………………………………..III
目錄……………………………………………………………………...V
緒論………………………………………………………………………1
藥品與儀器…………………………………………………………...….9
材料與方法
I. pGEX-2T表現系統之構築………………………………………. 15
II. GST-MCP-1融合蛋白之誘發表現與純化…………………….…. 23
III. GST-MCP-1融合蛋白的功能測定…………………….………….30
IV. 統計分析…………………………………………………..….…….36
結果…………………………………………………………….………. 37
討論…………………………………………………………………….46
參考文獻……………………………………………………………… ..52
圖…………………………………………………………….………… .60
附錄………………………………………………….………………… .76

Adams DH and Lloyd AR. (1997) Chemokines: leucocyte recruitmemt and activation cytokines. Lancet 349: 490-495,
Anand-Apte B, Zetter BR, Viswanathan A, Qiu RG, Chen J, Ruggieri R, and Symons M. (1997) Platelet—derived growth factor and fibronectin-stimulated migration are differentially regulated by Rac and extracellular signal-regulated kinase pathways. J Biol Chem. 272: 30688-30692
Ashida N, Arai H, Yamasaki M and Kita T. (2001) Distinct signaling pathways for MCP-1-dependent integrin activation and chemotaxis. J Biol Chem. 276: 16555-16560
Barnes MJ and Farndale RW. (1999) Collagens and atherosclerosis. Exp Gerontol. 34: 513-525
Battegay EJ, Raines EW, Seifert RA, Bowen-Pope DF, Ross R. (1990) TGF- induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell 63: 515-524
Ben-Baruch A, Michiel DF, Oppenheim JJ. (1995) Signals and receptors involved in recruitment of inflammatory cells. J Biol Chem. 270: 11703-11706
Bennett AM, Tonks NK. (1997) Regulation of distinct stages of skeletal muscle differentiation by mitogen-activated protein kinases. Science 278: 1288—1291
Boring L, Gosling J, Cleary M and Charo IF. (1998) Decreased lesion formation in CCR2-\- mice reveals a role for chemokines in initiation of atherosclerosis. Nature 394: 894-897
Brunet A and Pouyssegur, J (1996) Identification of MAP kinase domains by redirecting stress signals into growth factor responses. Science 272: 1652—1655
Camblen B, Pomeranz M, Milllet MA, Rossi B and Schmid-alliana A. (2001) Signal transduction involved in MCP-1-mediated monocytic transendothelial migration. Blood 97: 359-366,
Chen YL, Chang YJ and Jiang MJ. (1999) Monocyte chemotactic protein-1 gene and protein expression in atherogenesis of hypercholesterolemic rabbits. Atherosclerosis 143:115-123
Crawley JB, Rawlinson L, Lali FV, Page TH, Saklatvala J and Foxwell BM. (1997) T cell proliferation in response to interleukins 2 and 7 requires p38 MAP kinase activation. J Biol Chem. 272: 15023—15027
Cushing SD, Berliner JA, Valente AJ, Territo MC, Navab M, Parhami F, Gerrity R, Schwartz CJ and Fogelman AM. (1990) Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc Natl Acad Sci U S A. 1990 87: 5134-8.
Derijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ and Davis RJ. (1995) Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science. 267: 682-5.
Foltz IN, Lee JC, Young PR and Schrader JW (1997) Hemopoietic growth factors with the exception of interleukin-4 activate the p38 mitogen-activated protein kinase pathway. J Biol Chem. 272: 3296—3301
Fuentes ME, Durham SK, Swerdel MR, Lewin AC, Barton DS, Megill JR, Bravo R and Lira SA. (1995) Controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1. J Immunol. 155: 5769-5776.
Furukawa Y, Matsumori A, Ohashi N, Shioi T, Ono K, Harada A, Matsushima K and Sasayama S. (1999) Anti—Monocyte Chemoattractant Protein-1/Monocyte Chemotactic and Activating Factor Antibody Inhibits Neointimal Hyperplasia in Injured Rat Carotid Arteries. Circ Res. 84: 306-314
Grewal IS, Rutledge BJ, Fiorillo JA, Gu L, Gladue RP, Flavell RA and Rollins BJ. (1997) Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1. J Immunol. 159: 401-8.
Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK,Libby P and Rollins BJ. (1998) Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol cell 2: 275-281.
Gunn MD, Nelken NA, Liao X and Williams LT. (1997) Monocyte chemoattractant protein-1 is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation. J Immunol. 158:376-83.
Hall A. (1998) Rho GTPases and the actin cytoskeleton. Science 279:509-514.
Han J, Lee JD, Bibbs L and Ulevitch RJ. (1994) A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science. 265: 808-11.
Hayes IM, Jordan NJ, Tower S, Smith G, Paterson JR, Earnshaw JJ, Roach AG, Westwick J and Williams RJ. (1998) Human vascular smooth muscle cells express receptors for CC chemokines. Atherioscler Thromb Vasc Biol. 18: 397-403
Hedges JC, Dechert MA, Yamboliev IA, Martin JL, Hickey E, Weber LA and Gerthoffer WT. (1999) A role for p38 (MAPK)/HSP27 pathway in smooth muscle cell migration. J Biol Chem. 274: 24211-9.
Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, Takagi M, Matsumoto K, Miyazono K and Gotoh Y. (1997) Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science. 275: 90-4.
Ikeda U, Okada K, Ishikawa S-E, Saito T, Kashara T and Shimada K. (1995) Monocyte chemoattractant protein 1 inhibits growth of rat vascular smooth muscle cells. Am J Physiol. 268: H1021-H1026
Jarnagin k, Grunberger D, Mulkins M, Wong B, Hemmerich S, Paavola C., Bloom A, Bhakta S., Diehl F, Freedman R, McCarley D, Polsky I, Ann PT, Kosaka A and Handel TM. (1999) Identification of surface residues of the monocyte chemotactic protein 1 that affect signaling through the receptor CCR2. Biochemistry. 38: 16167-16177
Jones RD, Hancock JT and Morice AH. (2000) NADPH oxidase: a universal oxygen sensor. Free Radic Biol Med. 29: 416-24.
Libby P and O’Brien KV. (1984) The role of protein breakdown in growth, quienscence, and starvation of vascular smooth muscle cells. J cell. Physiol. 118:317-323
Lin A, Minden A, Martinetto H, Claret FX, Lange-Carter C, Mercurio F, Johnson GL and Karin M. (1995) Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2. Science. 268: 286-90.
Leeuwen RTJ. (1996) Extracellular proteolysis and the migrating vascular smooth muscle cell. Fibrinolysis 10:59-74
Mitchell R, McCulloch D, Lutz E, Johnson M, MacKenzie C, Fennell M, Fink G, Zhou W and Sealfon SC. (1998) Rhodopsin-family receptors associate with small G proteins to activate phospholipase D. Nature 392: 411-414
Nelken NA, Coughlin SR, Gordon D and Wilcox JN. (1991) Monocyte chemoattractant protein-1 in human atheromatous plaques. J Clin Invest. 88: 1121-7.
Nguyen DHD, Catling AD, Webb DJ, Sankovic M, Walker LA, Somlyo AV, Weber MJ and Gonias SL. (1999) Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner. J Cell Biol. 12: 149-164
Nishio E and Watanabe Y. (1997) The involvement of reactive oxygen species and arachidonic acid in alpha 1-adrenoceptor-induced smooth muscle cell proliferation and migration. Br J Pharmacol. 121: 665-70.
Patterson C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, Horaist C, Ballinger CA, Brasier AR, Bode C and Runge MS. (1999) Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo. J Biol Chem. 274: 19184-19822
Piotrowicz RS and Levin EG. (1997) Basolateral membrane-associated 27-kDa heat shock protein and microfilament polymerization. J Biol Chem. 272: 25920-25927
Porreca E, Febbo CD, Reale M, Casytellani ML, Baccante G, Barbacane R, Conti P, Cuccurullo F and Poggi A. (1997) Monocyte chamotactic protein 1 (MCP-1) is a moitogen for cultured rat vascular smooth muscle cells. J Vasc Res. 34:58-65
Rollins BJ, Yoshimura T, Leonard EJ and Pober JS. (1990) Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE. Am J Pathol. 136:1229-33
Rosenfeld ME. (1991) Oxidase LDL affects multiple atherogenic cellular responses. Circualtion 83:2137-2140
Ross R. (1999) Atherosclerosis-an inflammatory disesease. N. Eng. J Med. 340: 115-123,
Sadayama S, Okada M and Matsumori A. (2000) Chemokines and cardiovascular diseases. Cardiovasc Res. 45: 167-169,
Salcedo R, Ponce ML, Young HA, Wasserman K, Ward JM, Kleinman HK, Oppenheim JJ and Murphy WJ. (2000) Human endothelial cells express CCR2 and respond to MCP-1: direct role of MCP-1 in angiogenesis and tumor progression. Blood. 96: 34-40.
Streblow DN, Soderberg-Naucler C, Vieira J, Smith P, Wakabayashi E, Ruchti F, Mattison K, Altschuler Y and Nelson JA. (1999) The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration. Cell. 99: 511-20.
Strieter RM, Wiggins R, Phan SH, Wharram BL, Showell HJ, Remick DG, Chensue SW and Kunkel SL. (1989) Monocyte chemotactic protein gene expression by cytokine-treated human fibroblasts and endothelial cells. Biochem Biophys Res Commun.162: 694-700.
Sundaresan M, Yu ZX, Ferrans VJ, Irani K and Finkel T. (1995) Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270: 296-299
Seasholtz TM, Majumdar M, Kaplan DD and Brown JH. (1999) Rho and Rho kinase mediate thrombin-stimulated vascular smooth muscle cell DNA synthesis and migration. Circ Res. 84: 1186-93.
Tamura M, Kai T, Tsunawaki S, Lambeth JD and Kameda K. (2000) Direct interaction of actin with p47(phox) of neutrophil NADPH oxidase. Biochem Biophys Res Commun.276: 1186-90.
Thyberg J, Hedin U, Sjolund M, Palmberg L and Bottger BA. (1990) Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis 10: 966-990
Valente AJ, Graves DT, Vialle-Valentin CE and Delgado R. (1988) Purification of a monocyte chemotactic factor secreted by nonhuman primate vascular cells in culture. Biochemistry 27: 4162-4168
Wang JM, Sica A, Peri G, Walter S, Padura IM, Libby P, Ceska M, Lindley I, Colotta F and Mantovani A. (1991) Expression of monocyte chemotactic protein and interleukin-8 by cytokine-activated human vascular smooth muscle cells. Arterioscler Thromb. 11:1166-74.
Xia Z, Dickens M, Raingeaud J, Davis RJ and Greenberg ME. (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 1326—1331
Yen H., Zhang Y., Penfold S. and Rollins BJ. (1997) MCP-1 mediated chemotaxis requires actiovation of non-overlapping signal transduction pathways. J Leukoc Biol. 61:529-532
Yla-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshimura T, Leonard EJ, Witztum JL and Steinberg D. (1991) Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci U S A. 88: 5252-6.
Yoshimura T, Robinson EA, Tanaka S, Appella E, Kuratsu JI and Leonard EJ. (1989) Purification and amino acid analysis of two human glioma-derived monocyte chemoattractants. J Exp Med. 169:1449-1459
Yu X, Dluz S, Graves DT, Zhang L, Antoniades HN, Hollander W, Prusty S, Valente AJ, Schwartz CJ and Sonenshein GE. (1992) Elevated expression of monocyte chemoattractant protein 1 by vascular smooth muscle cells in hypercholesterolemic primates. Proc Natl Acad Sci U S A. 89: 6953-7.